CN105693804B - The method and its application of ergosterol are extracted from Armillaria luteo-virens - Google Patents
The method and its application of ergosterol are extracted from Armillaria luteo-virens Download PDFInfo
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Abstract
The present invention provides a kind of method that ergosterol is extracted from Armillaria luteo-virens：Extracted acetone cold soaking is added after the sub- ultramicro grinding of Armillaria luteo-virens, filter to take supernatant and be concentrated under reduced pressure；In Silica normal phase silica gel chromatography post separations after dissolving, using A phases n-hexane, B phase ethyl acetate binary mobile phases, eluted by 100%0~15min of A phases, 60%15~35min of A phases, 100%35~60min of B phases, collect 25 29min eluents and be concentrated under reduced pressure；Using A phases n-hexane, B phase ethyl acetate binary mobile phases, eluted by the 45min of A phases 88%0 in Silica normal phase silica gel chromatography post separations again after dissolving, collect 27.5 36min eluent rotary evaporations, obtain purity up to more than 95% ergosterol.The separation method is stable, and repeatability is high, and preparation amount is big, and obtained ergosterol can be used for preparing in medicines resistant to liver cancer.
Description
Technical field
The present invention relates to technical field of extraction of Chinese traditional medicine, and in particular to a kind of side that ergosterol is extracted from Armillaria luteo-virens
Method, and application of the obtained ergosterol in medicines resistant to liver cancer is prepared.
Background technology
Ergosterol (ergosterol), also known as ergosterol, are a kind of important phytosterols, are primarily present in yeast
In ergot.Ergosterol is the precursor for producing calciferol, plays the role of calciferol, is also the important of microbial cell film
Component part, integrality, the activity of membrane bound enzyme, the mobility of film, cell viability and cellular material to ensuring cell membrane
Transport etc. plays an important role.
Research shows that ergosterol cannot be only used for the production of the medicines such as flavonoids, and ergosterol is with physiology
The safe natural drug of activity application, is also the growth hormone of animals and plants, with anti-inflammatory, bring down a fever, antiulcer action, for treatment palace
The tumor diseases such as neck cancer, cutaneum carcinoma have obvious curative effects.Ergosterol can also prevent the dry skins such as vola, knee and palm and,
It can prevent and suppress corn to be formed, improve skin feel, be cosmetics, the masters of skin care item such as production perfume, shampoo, skin lotion
Want one of active component.
Publication No. CN102659890B Chinese invention patent is authorized to disclose a kind of extraction of ergosterol in university degree
Method, it take extraction, concentration, cracking, close ester extractions, saponification, urge extraction and the series of steps such as purify after successfully extract
Ergosterol, but this method not only complex steps, time-consuming, and its obtained ergosterol purity can only achieve 80%, this
Also there is gap as the purity requirement of drug component for ergosterol.
The content of the invention
The technical problems to be solved by the invention be for above prior art problem there is provided one kind from Armillaria luteo-virens
The method for extracting ergosterol, this method is isolated and purified using two-dimensional highly effective liquid phase chromatographic, and separation method is stable, repeatability
Height, preparation amount is big, and simple to operate time saving, obtained ergosterol purity can reach more than 95%.
The technical solution adopted in the present invention is：
A kind of method that ergosterol is extracted from Armillaria luteo-virens, this method comprises the following steps：
(1) using Armillaria luteo-virens fructification as raw material, room temperature is dried in the shade after processing, the drying and dewatering at a temperature of 40~60 DEG C,
Then ultramicro grinding processing is carried out, Ultramicro-powder is obtained；
(2) Ultramicro-powder is added into analysis pure water by 1g ︰ (6~15) mL solid-liquid ratio, in 60~90 DEG C of temperature after being well mixed
Degree is lower to extract 1~2h, and extraction is centrifuged after terminating under 4000~8000rpm rotating speeds, takes precipitation to repeat 1~3 time, it is heavy to finally obtain
Shallow lake is put into 50~80 DEG C of drying process of baking oven；
(3) the acetone cold soaking for adding 1~5 times by mass volume ratio (g/mL) in drying precipitated obtained by step (2) is carried
Take 3~8h, extraction to be centrifuged after terminating under 4000~8000rpm rotating speeds, take precipitation to repeat 1~3 time, collect all supernatants and subtract
Pressure is concentrated into paste, obtains small molecular extract；
(4) n-hexane-ethyl acetate that quality percent by volume (g/mL) adds 5~10 times is pressed in small molecular extract
Binary mixed solvent, carries out ultrasonic dissolution, and excessively organic filter membrane after dissolving obtains the one-dimensional loading containing small molecular extract molten
Liquid；
(5) high performance liquid chromatography separation is carried out to one-dimensional load solution：The chromatographic column used is Silica purification on normal-phase silica gel color
Post is composed, the mobile phase used is two end number mixing organic phase, and wherein A phases are n-hexane, and B phases are ethyl acetate, and sample size is 800-
1200mL/ pins, flow rate of mobile phase is 600-750mL/min, detector is UV-detector, Detection wavelength 260nm, and use is washed
Off-square formula is the 0~15min of isocratic elution of A phase concentrations 100%, A phase concentrations 60% (i.e. by percent by volume A phases 60%, B phases
40% proportioning form, under similarly) 15~35min of isocratic elution, the 35~60min of isocratic elution of B phase concentrations 100%, according to ultraviolet
Absorption spectrum collects 25-29min eluents (Fr6 peaks) as purpose component, is concentrated under reduced pressure into paste, obtains one-dimensional liquid phase group
Point；
(6) 5~10 times of n-hexane-second is added by quality percent by volume (g/mL) in obtained one-dimensional liquid phase component
Acetoacetic ester binary mixed solvent, carries out ultrasonic dissolution, excessively organic filter membrane, obtains two-dimentional load solution after dissolving；
(7) two-dimensional highly effective liquid phase chromatographic separation is carried out：The chromatographic column used for Silica normal phase silica gel chromatography posts, use
Mobile phase is two end number mixing organic phase, and wherein A phases are n-hexane, and B phases are ethyl acetate, and sample size is 20-80mL/ pins, flowing
Phase flow velocity is 40-80mL/min, and detector is UV-detector, Detection wavelength 260nm；The type of elution used is A phase concentration
88% isocratic elution 0-45min, collects 27.5-36min eluents (Fr6-4 peaks) according to ultra-violet absorption spectrum and is used as purpose group
Point, rotary evaporation is concentrated to dryness, as the object of the invention compound ergosterol.
Preferably, in the step (4), (6) in n-hexane-ethyl acetate binary mixed solvent n-hexane volume integral
Number is 50~90%.
Preferably, the size of the Silica normal phase silica gel chromatography posts in the step (5) is diameter 50mm, column length
250mm, wherein silicon ball particle diameter are 8 μm, and column temperature is room temperature or 25-40 DEG C when using.
Preferably, the size of the Silica normal phase silica gel chromatography posts in the step (7) is diameter 50mm, column length
250mm, wherein silicon ball particle diameter are 10 μm, and column temperature is room temperature or 25-40 DEG C when using.
The ergosterol monomeric compound obtained the present invention further provides said extracted is in medicines resistant to liver cancer is prepared
Using.The ergosterol monomeric compound is as active ingredient, according to a conventional method such as oral, injection, with being subjected to pharmaceutically
Carrier or excipient, are made the formulation for being adapted to the administering modes such as oral, injection, such as：Tablet, capsule, injection etc..
According to the understanding of those of ordinary skill in the art, the dosage of ergosterol should according to age for the treatment of target,
It typically can be 200-300mg/ days depending on the factors such as body weight, disease severity.
Because the material Polarity comparision in raw material Armillaria luteo-virens of the present invention is concentrated, so the present invention selects two-dimensional highly effective liquid
Phase chromatogram carries out the extraction of active principle.
First, in the condition selection of one-dimensional separation：Test of many times result shows that Silica normal phase silica gel chromatographies post is to institute
Extract matter separating effect is preferable, and because Armillaria luteo-virens acetone extract polarity is less than normal, in thick oily thing, during sample dissolution
Through testing n-hexane-ethyl acetate binary mixed solvent (n-hexane volume fraction 50~90%) favorable solubility, therefore selection
N-hexane, ethyl acetate are two end number mixing mobile phase, and by investigating after different types of elution, flow velocity and sample size, are as a result shown
Show that " 0~15min of isocratic elution of A phase concentrations 100%, the 15~35min of isocratic elution of A phase concentrations 60%, B phases are dense using the present invention
Best results are separated during the type of elution for spending 100% 35~60min " of isocratic elution, and sample size is in 800-1200mL/ pin scopes
Repeatability is good when interior, mobile phase is in the range of 600-750mL/min.
Secondly, in the condition selection of two dimensional separation：Silica normal phase silica gel chromatography posts are reused, in elution requirement selection
Impurity that can be by small part polarity with ergosterol relatively during for A 88% isocratic elutions of phase concentration is separated, and obtains purity
Up to 95% ergosterol monomeric compound.
Generally speaking, the method technique of present invention extraction ergosterol monomeric compound from Armillaria luteo-virens is simple, when
Between the cycle it is short, reagent consumption is small, and preparation amount is big, and stably and controllable, reproducible, batch uniformity is higher, obtained ergot steroid
Alcohol purity is high, and stability is good, can be directly used for carrying out the subsequent applications such as medicine preparation.
Brief description of the drawings
Shown in Fig. 1 is the one-dimensional high performance liquid chromatography for the method that the present invention extracts ergosterol from Armillaria luteo-virens
Figure, wherein it is that embodiment 2, (c) are embodiment 3 that (a), which is embodiment 1, (b),；
Shown in Fig. 2 is the two-dimensional highly effective liquid phase chromatographic for the method that the present invention extracts ergosterol from Armillaria luteo-virens
Figure, wherein it is that embodiment 2, (c) are embodiment 3 that (a), which is embodiment 1, (b),；
Shown in Fig. 3 is 1D1CNMR spectrum (MeOD, the 400MHz) data for the purpose component that the present invention is obtained；
Shown in Fig. 4 is 1D13HNMR spectrum (MeOD, the 400MHz) data for the purpose component that the present invention is obtained；
Shown in Fig. 5 is the suppression hepatoma cell proliferation figure for each component that the chromatographic isolation of the embodiment of the present invention 1 is obtained；
Shown in Fig. 6 is that the purpose component that the embodiment of the present invention 1 is obtained suppresses liver cancer cells increasing under the conditions of various concentrations
Grow figure；
Shown in Fig. 7 is that cell gives the cellular morphology figure after Fr6-4 in present example 4；
Shown in Fig. 8 is the structural formula of the object of the invention product ergosterol.
Embodiment
The present invention is further described in detail with reference to embodiments.It is noted that detailed description below is all to illustrate
Property, it is intended to provide further instruction to the present invention.Unless otherwise indicated, all scientific and technical terms that the present invention is used
With the identical meanings being generally understood that with the technical field of the invention personnel.
1st, raw material, material：
The harvesting of Armillaria luteo-virens fructification is from Qinghai Qilian and by identification；Hep-G2 cells are by Chinese Academy of Medical Sciences's blood
Ye Bing hospitals provide.
2nd, reagent：
Analysis pure water is produced by Mi Libo pure water meters；Chromatogram grade acetone, ethyl acetate, n-hexane are public purchased from Honeywell
Department；DMSO is purchased from Tianjin Concord Technology Co., Ltd.；F-12K culture mediums are purchased from Gibco companies.
3rd, instrument and equipment：
Micronizer is purchased from Sanqing Stainless Steel Apparatus Co., Ltd., Shandong；Tabletop refrigerated centrifuge is public purchased from Thermo
Department；Constant temperature blast drying oven is purchased from Shanghai Yiheng Scientific Instruments Co., Ltd；Rotary Evaporators Shanghai Ya Rong biochemical instruments factory；
Silica silica gel chromatographic columns are purchased from Beaune Ai Jier Co., Ltds；Preparative high performance liquid chromatography instrument has purchased from Jiangsu Chinese nation science and technology
Limit company；Cell real-time monitor is purchased from ACEABiosciences Inc (Essen Biology scientific company)；Cell culture incubator is purchased from
RS Biotech companies of Britain.
Embodiment 1：
The method that the present embodiment extracts ergosterol from Armillaria luteo-virens, comprises the following steps：
(1) using Armillaria luteo-virens fructification as raw material, room temperature is dried in the shade after processing, the drying and dewatering at a temperature of 60 DEG C, then-
20 DEG C of ultramicro grinding 5min, produce Armillaria luteo-virens fructification Ultramicro-powder.
(2) take above-mentioned Armillaria luteo-virens fructification Ultramicro-powder 1Kg, add 6.0L analysis pure water and extract 3 times at 90 DEG C, every time
1h, and collection supernatant respectively and precipitation are centrifuged under 8000rpm, it is repeated 3 times, gained precipitation is put at 60 DEG C of dryings of baking oven
Reason.
(3) 5h is extracted in the above-mentioned drying precipitated middle analysis grade acetone cold soaking for adding 2 times of volumes, and in 4000rpm rotating speeds
Under supernatant is collected by centrifugation, be concentrated under reduced pressure into paste, that is, obtain Armillaria luteo-virens fructification small molecular extract.
(4) mixed solvent of chromatographic grade n-hexane-chromatogram level ethyl acetate composition of 5 times of volumes is added by above-mentioned small molecule
Extract carries out ultrasonic dissolution, and n-hexane ratio of the total volume is 50%.0.45 μm of organic filter membrane is crossed after dissolving, obtains yellowish green
The one-dimensional sample solution of halimasch fructification small molecular extract.
(5) the one-dimensional sample solution carries out SPE using chromatogram is prepared.Filler uses particle diameter for 40 μm of positive
Separation material Silica particle diameter is 40 μm, and the separation chromatography column diameter used is 300mm for 100mm, column length, uses A for just
Hexane, B carry out SPE for the binary-mobile phase system of ethyl acetate；The condition of SPE is：3 times of column volume acetic acid second
Ester activates chromatographic column, 3 times of column volume n-hexanes balance, the sample solution loading containing small molecular extract, 3 times of column volumes just oneself
Alkane is eluted, the 5 times of n-hexane-ethyl acetate solution of column volume 50% elutions, obtains pre-treatment sample.
By the pre-treatment sample of acquisition enterprising using filler as positive separation material Silica (8 μm of particle diameter) preparation chromatogram
Row is isolated and purified, and the separation chromatography column diameter used is 250mm for 50mm, column length, and it is ethyl acetate two for n-hexane, B to use A
First flow visualizing is isolated and purified；The condition isolated and purified is：0~15min 100%A, 0%B, 15~35min 60%
A, 40%B, 35~50min 0%A, 100%B, according to gradient elution sample, Detection wavelength is 260nm, and sample size is 1.2g,
Flow velocity 700mL/min, is collected according to chromatographic peak, shown in chromatographic peak such as Fig. 1 (a), collects 25-29min eluents (Fr6 peaks)
As purpose component, paste is concentrated under reduced pressure into, one-dimensional liquid phase component is obtained.
(6) the n-hexane-ethyl acetate binary mixed solvent of 5 times of volumes is added in obtained one-dimensional liquid phase component, is entered
Row ultrasonic dissolution, crosses 0.45 μm of organic filter membrane, obtains two-dimentional load solution after dissolving.
(7) isolated and purified on using filler as positive separation material Silica preparation chromatogram, the positive separation of use
Material Silica particle diameter is 10 μm, and the separation chromatography column diameter used is 250mm for 50mm, column length, uses A for n-hexane, B
Isolated and purified for ethyl acetate binary-mobile phase system；The condition isolated and purified is：0~50min 88%A, 50~
55min 88%A~0%A, 55~60min 100%B, according to gradient elution sample, Detection wavelength：260nm, applied sample amount：
348mg, flow velocity 50mL/min.It is collected according to chromatographic peak, shown in chromatographic peak such as Fig. 2 (a), collects 27.5-36min eluents
(Fr6-4 peaks), as purpose component, rotary evaporation is concentrated to dryness, as the object of the invention component, is analyzed through HPLC, its purity reaches
To 99.2%.
NMR carbon spectrum hydrogen analysis of spectrum is carried out to purpose component obtained above, as a result as shown in Figure 3, Figure 4, it is wheat to show it
Angle sterol, its structural formula is as shown in Figure 8.
Embodiment 2：
The method that the present embodiment extracts ergosterol from Armillaria luteo-virens, comprises the following steps：
(1) Armillaria luteo-virens fructification room temperature is dried in the shade after processing, in 40 DEG C of drying and dewaterings, then in -10 DEG C of ultramicro grindings
5min, produces Armillaria luteo-virens fructification Ultramicro-powder.
(2) take above-mentioned Armillaria luteo-virens fructification Ultramicro-powder 1Kg, add 10.0L analysis pure water and extract 3 times at 80 DEG C, often
Secondary 1h, and collection supernatant respectively and precipitation are centrifuged under 5000rpm, it is repeated 3 times.Gained supernatant is concentrated under reduced pressure into paste storage
Deposit, gained precipitation is put into 70 DEG C of drying process of baking oven after merging.
(3) 5h is extracted in the above-mentioned drying precipitated middle analysis grade acetone cold soaking for adding 3 times of volumes, and in 5000rpm rotating speeds
Under supernatant is collected by centrifugation, be concentrated under reduced pressure into paste, that is, obtain Armillaria luteo-virens fructification small molecular extract.
(4) the chromatographic grade n-hexane of 8 times of volumes, chromatogram level ethyl acetate are added above-mentioned small molecular extract is subjected to ultrasound
Dissolving, n-hexane ratio of the total volume is 80%.0.45 μm of organic filter membrane is crossed after dissolving, Armillaria luteo-virens fructification is obtained small
The sample solution of molecule extractive.The sample solution carries out SPE using chromatogram is prepared.Filler uses particle diameter for 40 μm
Positive separation material Silica particle diameter be 40 μm, the separation chromatography column diameter used is 300mm for 100mm, column length, use
The binary-mobile phase system that A is n-hexane, B is ethyl acetate carries out SPE；The condition of SPE is：3 times of column volumes
Ethyl acetate activates chromatographic column, 3 times of column volume n-hexane balances, the sample solution loading containing small molecular extract, 3 times of cylinders
Product n-hexane elution, the 5 times of n-hexane-ethyl acetate solution of column volume 50% elutions, obtains pre-treatment sample.
(5) by the pre-treatment sample of acquisition in the preparation chromatogram using filler as positive separation material Silica (8 μm of particle diameter)
Upper to be isolated and purified, the separation chromatography column diameter used is 250mm for 50mm, column length, and it is acetic acid second for n-hexane, B to use A
Ester binary-mobile phase system is isolated and purified；The condition isolated and purified is：0~15min 100%A, 0%B, 15~35min
60%A, 40%B, 35~50min 0%A, 100%B, according to gradient elution sample, Detection wavelength is 260nm, and sample size is
1.2g, flow velocity 650mL/min, is collected according to chromatographic peak, shown in chromatographic peak such as Fig. 1 (b), collects 25-29min eluents
(Fr6 peaks) is concentrated under reduced pressure into paste as purpose component, obtains one-dimensional liquid phase component.
(6) one-dimensional liquid phase component is carried out to separation on using filler as positive separation material Silica preparation chromatogram pure
Change, the positive separation material Silica used particle diameter is 10 μm, and the separation chromatography column diameter used is for 50mm, column length
250mm, it is that ethyl acetate binary-mobile phase system is isolated and purified for n-hexane, B to use A；The condition isolated and purified is：0
~50min, 88%A；50~55min, 88%A~0%A；55~60min, 0%A, according to gradient elution sample, Detection wavelength：
260nm, applied sample amount：348mg, flow velocity 50mL/min.It is collected, shown in chromatographic peak such as Fig. 2 (b), collects according to chromatographic peak
27.5-36min eluents (Fr6-4 peaks) are as purpose component, and rotary evaporation is concentrated to dryness, as the object of the invention component, warp
HPLC is analyzed, and its purity reaches 98.6%.
NMR carbon spectrum hydrogen analysis of spectrum is carried out to purpose component obtained above, it is ergosterol to show it, and its structural formula is as schemed
Shown in 8.
Embodiment 3：
The method that the present embodiment extracts ergosterol from Armillaria luteo-virens, comprises the following steps：
(1) Armillaria luteo-virens fructification room temperature is dried in the shade after processing, in 50 DEG C of drying and dewaterings, then in -15 DEG C of ultramicro grindings
10min, produces Armillaria luteo-virens fructification Ultramicro-powder.
(2) take above-mentioned Armillaria luteo-virens fructification Ultramicro-powder 1Kg, add 15L analysis pure water and extract 3 times at 75 DEG C, every time
2h, and collection supernatant respectively and precipitation are centrifuged under 6000rpm, it is repeated 3 times.Precipitation is put at 80 DEG C of dryings of baking oven after merging
Reason.
(3) 8h is extracted in the above-mentioned drying precipitated middle analysis grade acetone cold soaking for adding 5 times of volumes, and in 6000rpm rotating speeds
Under supernatant is collected by centrifugation, be concentrated under reduced pressure into paste, that is, obtain Armillaria luteo-virens fructification small molecular extract.
(4) chromatographic grade n-hexane, the chromatogram level ethyl acetate for adding 10 times of volumes are surpassed above-mentioned small molecular extract
Sound dissolves, and n-hexane ratio of the total volume is 90%.0.45 μm of organic filter membrane is crossed after dissolving, Armillaria luteo-virens fructification is obtained
The sample solution of small molecular extract.The sample solution carries out SPE using chromatogram is prepared.Filler uses particle diameter for 40 μ
M positive separation material Silica particle diameter is 40 μm, and the separation chromatography column diameter used is 300mm for 100mm, column length, is adopted
SPE is carried out with the binary-mobile phase system that A is n-hexane, B is ethyl acetate；The condition of SPE is：3 times of cylinders
Product ethyl acetate activation chromatographic column, 3 times of column volume n-hexane balances, the sample solution loading containing small molecular extract, 3 times of posts
Volume n-hexane is eluted, the 5 times of n-hexane-ethyl acetate solution of column volume 50% elutions, obtains pre-treatment sample.
(5) by the pre-treatment sample of acquisition in the preparation chromatogram using filler as positive separation material Silica (8 μm of particle diameter)
Upper to be isolated and purified, the separation chromatography column diameter used is 250mm for 150mm, column length, and it is acetic acid for n-hexane, B to use A
Ethyl ester binary-mobile phase system is isolated and purified；The condition isolated and purified is：0~15min 100%A, 0%B, 15~
35min 60%A, 40%B, 35~50min 0%A, 100%B, according to gradient elution sample, Detection wavelength is 260nm, sample introduction
Measure as 1.2g, flow velocity 750mL/min, be collected according to chromatographic peak, shown in chromatographic peak such as Fig. 1 (c), collect 25-29min elutions
Liquid (Fr6 peaks) is concentrated under reduced pressure into paste, obtains one-dimensional liquid phase component as purpose component.
(6) one-dimensional liquid phase component is carried out to separation on using filler as positive separation material Silica preparation chromatogram pure
Change, the positive separation material Silica used particle diameter is 10 μm, and the separation chromatography column diameter used is for 50mm, column length
250mm, it is that ethyl acetate binary-mobile phase system is isolated and purified for n-hexane, B to use A；The condition isolated and purified is：0
~50min, 88%A~88%A；50~55min, 88%A~0%A；55~60min, 0%A~0%A, according to gradient elution
Sample, Detection wavelength：260nm, applied sample amount：348mg, flow velocity 50mL/min.It is collected according to chromatographic peak, chromatographic peak such as Fig. 2
(c) shown in, 27.5-36min eluents (Fr6-4 peaks) are collected as purpose component, rotary evaporation is concentrated to dryness, is the present invention
Purpose component, is analyzed through HPLC, and its purity reaches 97.3%.
NMR carbon spectrum hydrogen analysis of spectrum is carried out to purpose component obtained above, it is ergosterol to show it, and its structural formula is as schemed
Shown in 8.
Embodiment 4：Resisting liver cancer activity is tested
(1) in 5%CO2, 37 DEG C, under saturated humidity, with F-12K (10%FBS+1%PS) medium culture human liver cancer
HepG2 cells, the cell for choosing logarithmic phase growth is used as experimental cell.It is diluted to after cell count with culture medium certain density
Cell suspension.
(2) cell real-time monitor is put into 5%CO2, in 37 DEG C of saturated humidity incubators.8 orifice plates are taken, are added per hole
150 μ L F-12K culture mediums, are put into cell real-time monitor and walk baseline, cover taking-up octal plate after baseline, are added per hole dilute
The μ L of HepG2 cell suspensions 345 released, to every hole cell number about 20,000, stand 3min, and cell is observed under inverted microscope is
It is no uniform.Add the samples that have diluted of 5 μ L, sample three concentration of setting per hole, final concentration be respectively 20 μ g/ml, 50 μ g/ml,
80μg/ml.With the culture medium containing 0.1%DMSO as blank control group, it is put into cell real-time monitor and detects, obtain yellowish green
The real-time vegetative map of halimasch fructification resisting liver cancer activity, experimental result is as shown in Figure 5, Figure 6.Experimental result illustrates Armillaria luteo-virens
Fructification component Fr6-4, i.e. ergosterol have good inhibiting effect to the propagation of liver cancer cells, and thin to liver cancer HepG2
The value-added inhibition of born of the same parents and the concentration of ergosterol are proportionate.Fig. 7 is that cell is given in 50 μ g/ml, 80 μ g/ml Fr6-4
State diagram afterwards, when the concentration of ergosterol is 80 μ g/ml, hepatoma Hep G 2 cells stop growing substantially.Therefore, ergot steroid
Alcohol has the potentiality for developing into medicines resistant to liver cancer.
The present invention carries out the monitoring of resisting liver cancer activity experiment using the real-time n cell analytical technologies of RTCA.RTCA is real-time
N cell analytical technology be it is a kind of set up based on new cytoanalyze there is real-time monitoring, high information quantity, nothing
Need the detection technique of the particular advantages such as mark, full-automatic, high sensitivity and high accuracy.The cytoanalyze is by being embedded in E-
On plate plates the microelectronics inductor impedance variations of bottom hole go the presence or absence of permissive cell and it is adherent, stick and extent of growth
Change.In cytotoxicity detection, can in real time, the intuitively cell biological such as reacting cells propagation, survival, apoptosis, metamorphosis
Learn change.The technology causes cell experiment to become more time-saving and efficiency.
The present embodiments relate to material, reagent and experimental facilities, be to meet traditional Chinese medicine extraction unless otherwise instructed
Commercially available prod.
It is described above, only the preferred embodiments of the present invention, it is noted that for those skilled in the art
For, on the premise of the core technology of the present invention is not departed from, improvements and modifications can also be made, these improvements and modifications also should
Belong to the scope of patent protection of the present invention.Any change in the implication and scope suitable with claims of the present invention, all
It is considered as being included within the scope of the claims.
Claims (4)
1. a kind of method that ergosterol is extracted from Armillaria luteo-virens, it is characterised in that comprise the following steps：
(1) using Armillaria luteo-virens fructification as raw material, room temperature is dried in the shade after processing, the drying and dewatering at a temperature of 40~60 DEG C, then
Ultramicro grinding processing is carried out, Ultramicro-powder is obtained；
(2) Ultramicro-powder is added into analysis pure water by 1g ︰ (6~15) mL solid-liquid ratio, after being well mixed at a temperature of 60~90 DEG C
1~2h is extracted, extraction centrifuges under 4000~8000rpm rotating speeds after terminating, takes precipitation to repeat 1~3 time, finally obtain precipitation and put
Enter 50~80 DEG C of drying process of baking oven；
(3) the acetone cold soaking for adding 1~5 times by mass volume ratio in drying precipitated obtained by step (2) extracts 3~8h, carries
Take and centrifuged after end under 4000~8000rpm rotating speeds, take precipitation to repeat 1~3 time, collect all supernatants and be concentrated under reduced pressure into cream
Shape, obtains small molecular extract；
(4) the n-hexane-ethyl acetate two end number mixing that 5~10 times of quality percent by volume addition is pressed in small molecular extract is molten
Agent, carries out ultrasonic dissolution, and excessively organic filter membrane after dissolving obtains the one-dimensional load solution containing small molecular extract；
(5) high performance liquid chromatography separation is carried out to one-dimensional load solution：The chromatographic column used for Silica normal phase silica gel chromatography posts,
The mobile phase used is two end number mixing organic phase, and wherein A phases are n-hexane, and B phases are ethyl acetate, and sample size is 800-
1200mL/ pins, flow rate of mobile phase is 600-750mL/min, detector is UV-detector, Detection wavelength 260nm, and use is washed
Off-square formula is the 0~15min of isocratic elution of A phase concentrations 100%, the 15~35min of isocratic elution of A phase concentrations 60%, B phase concentrations
100% 35~60min of isocratic elution, collects 25-29min eluents as purpose component according to ultra-violet absorption spectrum, depressurizes dense
Paste is reduced to, one-dimensional liquid phase component is obtained；
(6) 5~10 times of n-hexane-ethyl acetate binary is added by quality percent by volume in obtained one-dimensional liquid phase component
Mixed solvent, carries out ultrasonic dissolution, excessively organic filter membrane, obtains two-dimentional load solution after dissolving；
(7) two-dimensional highly effective liquid phase chromatographic separation is carried out：The chromatographic column used is Silica normal phase silica gel chromatography post, the flowing of use
It is mutually two end number mixing organic phase, wherein A phases are n-hexane, B phases are ethyl acetate, and sample size is 20-80mL/ pins, mobile phase stream
Speed is 40-80mL/min, and detector is UV-detector, Detection wavelength 260nm；The type of elution used is A phase concentration 88%
Isocratic elution 0-45min, 27.5-36min eluents are collected as purpose component, rotary evaporation concentration according to ultra-violet absorption spectrum
Extremely do, as the object of the invention compound ergosterol.
2. the method according to claim 1 that ergosterol is extracted from Armillaria luteo-virens, it is characterised in that：The step
(4), the volume fraction of n-hexane is 50~90% in n-hexane-ethyl acetate binary mixed solvent in (6).
3. the method according to claim 1 that ergosterol is extracted from Armillaria luteo-virens, it is characterised in that：The step
(5) size of the Silica normal phase silica gel chromatography posts in is diameter 50-150mm, column length 250mm, and wherein silicon ball particle diameter is 8 μm,
Column temperature is room temperature or 25-40 DEG C when using.
4. the method according to claim 1 that ergosterol is extracted from Armillaria luteo-virens, it is characterised in that：The step
(7) size of the Silica normal phase silica gel chromatography posts in is diameter 50mm, column length 250mm, and wherein silicon ball particle diameter is 10 μm, is used
Shi Zhuwen is room temperature or 25-40 DEG C.
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